Canine squamous cell carcinoma (SCC) shows highly invasive and locally destructive growth. In animal models and human cancer cases, periostin plays a critical role in the enhancement of cancer growth; however, the mechanism of involvement in canine cancers remains unknown. The aim of this study was to examine the involvement of periostin in the pathophysiology of SCC in dogs. We examined the localization of periostin and periostin-producing cells in 20 SCC and three squamous papilloma specimens. Furthermore, we focused on transforming growth factor (TGF)-β1, which was assumed to be an inducing factor of periostin, using culture cells. By immunohistochemistry, limited periostin expression in the stroma was observed in all squamous papillomas. In SCC, periostin protein diffusely expressed at the tumor invasion front of cancer growth. In situ hybridization revealed that periostin mRNA was expressed in the stromal fibroblasts in SCC. In vitro analysis determined that canine SCC cells expressed significantly higher levels of TGF-β1 mRNA compared with canine keratinocytes. In addition, recombinant TGF-β1 induced secretion of periostin from cultured dermal fibroblasts. These data suggest that periostin produced by stromal fibroblasts may be involved in the pathophysiology of canine SCC. TGF-β1 derived from SCC cells may stimulate fibroblasts to produce periostin.